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Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9.

CRISPR-Cas9 is now the leading method for genome editing and is advancing for the treatment of human disease. CRIPSR has promise in treating neurological diseases, but traditional viral-vector-delivery approaches have neurotoxicity limiting their use. Here we describe a simple method for non-viral transfection of primary human DRG (hDRG) neurons for CRISPR-Cas9 editing. We edited TRPV1, NTSR2, and CACNA1E using a lipofection method with CRISPR-Cas9 plasmids containing reporter tags (GFP or mCherry). Transfection was successfully demonstrated by the expression of the reporters two days post-adm…

Novel mechanism of cisplatin resistance in head and neck squamous cell carcinoma involving extracellular vesicles and a copper transporter system.

BACKGROUND: Cisplatin (CDDP) plays a central role in chemotherapy for head and neck squamous cell carcinoma (HNSCC), but drug resistance in HNSCC chemotherapy remains a problem, and the mechanism of CDDP resistance is unclear. We investigated CDDP-resistance mechanisms mediated by extracellular vesicles (EVs) and ATPase copper transporting beta (ATP7B) in HNSCC. METHODS: We established CDDP-resistant sublines of HNSCC cells and verified their ATP7B expression. We used an EV secretion inhibitor (GW4869) and ATP7B short hairpin (sh)RNA transfection to examine the correlation between EV secretio…

Ultrasound-targeted microbubble technology facilitates SAHH gene delivery to treat diabetic cardiomyopathy by activating AMPK pathway.

Diabetic cardiomyopathy (DCM) is a cardiovascular complication with no known cure. In this study, we evaluated the combination of ultrasound-targeted microbubble destruction (UTMD) and cationic microbubbles (CMBs) for cardiac S-adenosyl homocysteine hydrolase (SAHH) gene transfection as potential DCM therapy. Models of high glucose/fat (HG/HF)-induced H9C2 cells and streptozotocin-induced DCM rats were established. Ultrasound-mediated SAHH delivery using CMBs was a safe and noninvasive approach for spatially localized drug administration both in vitro and in vivo. Notably, SAHH overexpression …

Вирус везикулярного стоматита в качестве платформы для измерения активности протекционистов.

Ингибиторы протеинов являются одними из самых мощных антивирусных препаратов. Они успешно применяются против таких вирусов, как вирус иммунодефицита человека (ВИЧ), вирус гепатита С (ГХВ) и острый острый респираторный синдром коронавирус 2 (SARS-COV-2). Поэтому для выявления ингибиторов, способных стать антивирусными препаратами, важное значение имеют средства скрининга ингибиторов. В этой статье мы описываем недавно разработанные клеточные и вирусные платформы для проверки ингибиторов. Мы разработали методы, представленные здесь путем генетически модифицирующего вирус сосудистого стоматита, м

Совпадение в аннотации оригинала
Protease inhibitors are among the most powerful antiviral drugs. They have been used successfully against viruses, such as the human immunod … ocol: Mpro-On and -Off assay Alternate Protocol 1: Virus production with transient P- and L TransIT transfection Alternate Protocol 2: Virus production with transient P- and L Ca2PO4 transfection Alternate Protocol 3: Luciferase-based variation of the On assay Alternate Protocol 4: Screening assay with fluorescence-activated cell sorting readout Support Protoc …

Interleukin-24 Transduction Modulates Human Prostate Cancer Malignancy Mediated by Regulation of Anchorage Dependence.

BACKGROUND: Hormone therapy and chemotherapy are not effective for castrate-resistant prostate cancer, thus development of novel treatment strategies is required. Gene therapy involving transient high-copy transfection of interleukin (IL)-24 with an adenoviral vector can exert antitumor activity; however, the effects of stable IL-24 transfection are not fully understood. The aim of this study was to investigate the effects of IL-24 overexpression in prostate cancer cells, in vitro. MATERIALS AND METHODS: DU145 cells were transfected the IL-24 gene using a retroviral vector. Apoptosis inductio…

Lentiviral Transduction of Rat Schwann Cells and Dorsal Root Ganglia Neurons for In Vitro Myelination Studies.

Lentiviral transduction is a gene delivery method that provides numerous advantages over direct transfection and traditional retroviral or adenoviral delivery methods. It facilitates for the transduction of primary cells inherently difficult to transfect, delivers constructs of interest to nondividing as well as dividing cells, and permits the long-term expression of sizable DNA inserts (e.g., <7 kb). The study of peripheral nerve myelination at the molecular level has long benefited from the Schwann cells/dorsal root ganglia (DRG) neurons myelinating co-culture system. As this culture system …

miR-34a and its novel target, NLRC5, are associated with HPV16 persistence.

Persistent infection with human papillomavirus (HPV), particularly type 16, is causally associated with cervical cancer and its precursors. The role of miRNAs in HPV16 persistence currently remains unclear. Preliminary analysis of miRNA profile demonstrated that HPV16 infection caused a striking downregulation of miR-34a. Through bioinformatics analysis and dual-luciferase assay with site-directed mutagenesis strategy, NLRC5, a negative regulator of NF-κB signaling, was identified to be a novel interactor of miR-34a. Transfection of miR-34a mimic strikingly downregulated NLRC5 in the HPV16-pos…

A microRNA-152 that targets the phosphatase and tensin homolog to inhibit low oxygen induced-apoptosis in human brain microvascular endothelial cells.

Brain damage caused by perinatal asphyxia is dangerous for neonatal infants, but the mechanism by which it occurs remains elusive. In this s … d with miR-152 mimics, PTEN expression was inhibited at both the mRNA and protein levels. Moreover, transfection with the miR-152 mimic also inhibited apoptosis induced by hypoxia. Furthermore, expression of the pro-apoptotic gene Bax was downregulated while the anti-apoptotic gene Bcl2 was upregulated after miR-152 mimic transfection. Taken together, these re …

Unrevealing the role of P-protein on melanosome biology and structure, using siRNA-mediated down regulation of OCA2.

The pink-eyed dilution protein (P-protein) plays a critical role in melanin synthesis in melanocytes and retinal pigment epithelium cells. M … ed by the transmission electron microscopy. The P-protein inhibition was carried by P-protein-siRNA transfection to melan-a melanocytes, B16F10 mouse melanoma, and melan-p1 cells. Measurement of melanin contents, cellular tyrosinase, and different tyrosinase related proteins were also determined to investigate the effect of P-protein siRNA transfection on mela …

Knockdown of Cul4A increases chemosensitivity to gemcitabine through upregulation of TGFBI in lung cancer cells.

Cullin 4A (Cul4A) promotes oncogenesis through overexpression and then ubiquitination‑mediated proteolysis of tumor suppressors in various types of cancers. Transforming growth factor β‑induced (TGFBI) has been implicated as a tumor suppressor, which enhances gemcitabine chemosensitivity in lung cancer cells. The present study aimed to investigate the association of TGFBI and Cul4A and the mechanism by which Cul4A regulates TGFBI. In addition, we also evaluated the therapeutic value of Cul4A RNAi using adenoviral transfection of Cul4A RNAi in nude mouse xenograft models. We observed that knock…

MicroRNA-133b inhibits the growth of non-small-cell lung cancer by targeting the epidermal growth factor receptor.

Both the deregulation of microRNAs and epidermal growth factor receptor (EGFR) are emerging as important factors in non-small-cell lung cancer (NSCLC). Here, miR-133b was found to be associated with tumor stage, the extent of regional lymph node involvement, stage, visceral pleura or vessel invasion and EGFR mRNA expression in Chinese patients with NSCLC. Bioinformatic analysis and luciferase reporter assay revealed that miR-133b can interact specifically with the 3'-UTR of EGFR mRNA. Functionally, miR-133b transfection showed regulatory activity in translationally repressing EGFR mRNA. Moreov…

Apoptotic signaling induces hyperpermeability following hemorrhagic shock.

Hemorrhagic shock (HS) disrupts the endothelial cell barrier, resulting in microvascular hyperpermeability. Recent studies have also demonst … hondrial cytochrome c into the cytoplasm, and activation of caspase-3. This, along with the in vivo transfection of the proapoptotic peptide BAK (BH3), resulted in hyperpermeability (as visualized by intravital microscopy), release of mitochondrial cytochrome c into the cytoplasm, and activation of caspase-3. Conversely, transfection of the BAK (BH3) mutant ha …

Making V(D)J rearrangement visible: quantification of recombination efficiency in real time at the single cell level.

V(D)J recombination is of fundamental importance for the diversity of immunoglobulin and T cell receptor genes. An enhanced green fluorescent protein (EGFP) based assay was successfully developed to monitor V(D)J recombination efficiency. This assay makes V(D)J recombination visible at the single cell level in real time. Surprisingly, despite a high (60% to 90%) transfection efficiency, the EGFP based V(D)J recombination efficiency was found to be low ( approximately 1%) in 293 cells. The EGFP based V(D)J recombination efficiency correlated well with that achieved by the classical V(D)J recomb…

Protein-DNA binding and CpG methylation at nucleotide resolution of latency-associated promoters Qp, Cp, and LMP1p of Epstein-Barr virus.

Epstein-Barr viral (EBV) latency-associated promoters Qp, Cp, and LMP1p are crucial for the regulated expression of the EBNA and LMP transcripts in dependence of the latency type. By transient transfection and in vitro binding analyses, many promoter elements and transcription factors have previously been shown to be involved in the activities of these promoters. However, the latency promoters have only partially been examined at the nucleotide level in vivo. Therefore, we undertook a comprehensive analysis of in vivo protein binding and CpG methylation patterns at these promoters in five repr…

Expression of PPAR(alpha) in human hepatocytes and activation by trichloroacetate and dichloroacetate.

Dichloroacetate (DCA) and trichloroacetate (TCA) are carcinogenic metabolites of trichloroethylene (TCE), a known hepatocarcinogen in B6C3F1 … d human hepatocytes display PPAR(alpha) levels only slightly lower than cultured mouse hepatocytes. Transfection studies examining the endogenous hPPAR(alpha) activity revealed little or no receptor activation, even following treatment with high concentrations of peroxisome proliferators. In contrast human hepatocytes transfected with mPPAR(alpha) and mRXR(alp …

Cell surface GPI-anchoring of CD45 isoforms.

We have designed a new cell surface expression plasmid to study the structural and membrane-topological requirements for functioning of different isoforms of CD45, a leucocyte specific member of the protein tyrosine phosphatase (PTPase) family of proteins. Use of this vector in cell transfection experiments enabled us to produce multiple CD45 isoforms (ABC, B, Null), with their extracellular segment intact, and the entire membrane spanning and intracellular C-terminal domain replaced by a GPI-membrane-anchor and VSV-tag. Our strategy facilitated the identification and analysis of chimeric prot…

Molecular cloning of the human nucleotide-excision-repair gene ERCC4.

ERCC4 was previously identified in somatic cell hybrids as a human gene that corrects the nucleotide-excision-repair deficiency in mutant hamster cells. The cloning strategy for ERCC4 involved transfection of the repair-deficient hamster cell line UV41 with a human sCos-1 cosmid library derived from chromosome 16. Enhanced UV resistance was seen with one cosmid-library transformant and two secondary transformants of UV41. Cosmid clones carrying a functional ERCC4 gene were isolated from a library of a secondary transformant by selecting in Escherichia coli for expression of a linked neomycin-r…

Biological and biochemical characterization of an SV40-transformed xeroderma pigmentosum cell line.

We have isolated a subclone of the SV40-transformed xeroderma pigmentosum (XP) cell line SV40XP12RO. The cell line, designated M1, is highly … in the ability to induce SV40-DNA replication. The M1 subclone is an efficient recipient of DNA in transfection experiments. Transfection of these cells with the pSV2gpt plasmid shows that the M1 subclone is as efficient as the NIH 3T3 cell line in uptake and expression of foreign DNA. This cell line should be suitable for genetic analysis of the xeroderma pi …

Downregulation of FTL decreases proliferation of malignant mesothelioma cells by inducing G1 cell cycle arrest.

Pleural malignant mesothelioma is a malignant tumor with a poor prognosis that is strongly associated with asbestos exposure during its development. Because there is no adequate treatment for malignant mesothelioma, investigation of its molecular mechanism is important. The ferritin light chain (FTL) is a subunit of ferritin, and its high expression in malignant tumors, including malignant mesothelioma, has recently been reported; however, its role in malignant mesothelioma is unclear. The purpose of the present study was to clarify the function of FTL in malignant mesothelioma. The expression

TLR2, TLR3 and TLR5 regulation of pro-inflammatory and pro-labour mediators in human primary myometrial cells.

Preterm birth continues to be a significant global health care issue, due to our lack of understanding of the mechanisms that drive human la … ession of flagellin-induced cytokine and chemokine expression, ICAM-1 and MMP-9 expression. Lastly, transfection with these siRNAs decreased fsl-1, poly(I:C) and flagellin-induced NF-κB transcriptional activity. Our study signifies that these adaptor molecules are necessary for the proper production of cytokines, chemokines and pro-labour mediators after TLR l …